1. Field of the Invention
The present invention relates to biodegradable and biocompatible polymer compositions and more particularly to biodegradable copolymers and terpolymers; methods used for preparation of the recited polymers; articles useful for implantation or injection into the human body that are fabricated from said compositions; and, methods for the controlled release of biologically active substances.
2. Background
Biocompatible polymeric materials have been used extensively in therapeutic drug delivery and medical implant device applications. Sometimes, it is also desirable for such polymers to be, not only biocompatible, but also biodegradable to obviate the need for removing the polymer once its therapeutic value has been exhausted.
Conventional methods of drug delivery, such as frequent periodic dosing, are not ideal in many cases. For example, with highly toxic drugs, frequent conventional dosing can result in high initial drug levels at the time of dosing, often at near-toxic levels, followed by low drug levels between doses that can be below the level of their therapeutic value. However, with controlled drug delivery, drug levels can be more nearly maintained at therapeutic, but non-toxic, levels by controlled release in a predictable manner over a longer term.
If a biodegradable medical device is intended for use as a drug delivery or other controlled-release system, using a polymeric carrier is one effective means to deliver the therapeutic agent locally and in a controlled fashion, see Langer et al., Rev. Macro. Chem. Phys., C23(1), 61 (1983). As a result, less total drug is required, and toxic side effects can be minimized. Polymers have been used as carriers of therapeutic agents to effect a localized and sustained release. See Chien et al., Novel Drug Delivery Systems (1982). Such delivery systems offer the potential of enhanced therapeutic efficacy and reduced overall toxicity.
For a non-biodegradable matrix, the steps leading to release of the therapeutic agent are water diffusion into the matrix, dissolution of the therapeutic agent, and diffusion of the therapeutic agent out through the channels of the matrix. As a consequence, the mean residence time of the therapeutic agent existing in the soluble state is longer for a non-biodegradable matrix than for a biodegradable matrix, for which passage through the channels of the matrix, while it may occur, is no longer required. Since many pharmaceuticals have short half-lives, therapeutic agents can decompose or become inactivated within the non-biodegradable matrix before they are released. This issue is particularly significant for many bio-macromolecules and smaller polypeptides, since these molecules are generally hydrolytically unstable and have low permeability through a polymer matrix. In fact, in a non-biodegradable matrix, many bio-macromolecules aggregate and precipitate, blocking the channels necessary for diffusion out of the carrier matrix.
These problems are alleviated by using a biodegradable matrix that, in addition to some diffusional release, also allows controlled release of the therapeutic agent by degradation of the polymer matrix. Examples of classes of synthetic polymers that have been studied as possible biodegradable materials include polyesters (Pitt et al., Controlled Release of Bioactive Materials, (Baker, ed. 1980); polyamides; polyurethanes; polyorthoesters (Heller et al., Polymer Engineering Sci., 21:727 (1981); and polyanhydrides (Leong et al., Biomaterials 7:364 (1986). Specific examples of biodegradable materials that are used as medical implant materials are polylactide, polyglycolide, polydioxanone, poly(lactide-co-glycolide), poly(glycolide-co-polydioxanone), polyanhydrides, poly(glycolide-co-trimethylene carbonate), and poly(glycolide-co-caprolactone).
Polymers having phosphate linkages, called poly(phosphates), poly(phosphonates) and poly(phosphites), are known. The respective structures of these three classes of compounds, each having a different sidechain connected to the phosphorus atom, are as follows: 
The versatility of these polymers comes from the versatility of the phosphorus atom, which is known for a multiplicity of reactions. Its bonding can involve the 3p orbitals or various 3s-3p hybrids; spd hybrids are also possible because of the accessible d orbitals. Thus, the physico-chemical properties of the poly(phosphoesters) can be readily changed by varying either the R or Rxe2x80x2 group. The biodegradability of the polymer is due primarily to the physiologically labile phosphoester bond in the backbone of the polymer. By manipulating the backbone or the sidechain, a wide range of biodegradation rates are attainable.
An additional feature of poly(phosphoesters) is the availability of functional side groups. Because phosphorus can be pentavalent, drug molecules or other biologically active substances can be chemically linked to the polymer, as shown by Leong, U.S. Pat. Nos. 5,194,581 and 5,256,765. For example, drugs with xe2x80x94O-carboxy groups may be coupled to the phosphorus via an ester bond, which is hydrolyzable. The Pxe2x80x94Oxe2x80x94C group in the backbone also lowers the glass transition temperature of the polymer and, importantly, confers solubility in common organic solvents, which is desirable for easy characterization and processing.
Polylactide, referred to herein as PLA, and poly(lactide-co-glycolide), referred to herein as PLGA, are among the most popular and well characterized biodegradable polymeric materials used today for drug delivery and tissue engineering. Further, the present invention provides the ability to incorporate side chain modifications into both PLA and PLGA.
The growing needs in biomedical practice have continued to stimulate the studies for developing new biodegradable materials. Although several classes of synthetic polymers, including polyesters, poly(amino acid)s/polyamides, polyurethanes, poly(orthoester)s, poly(anhydride)s, polycarbonates, poly(imidocarbonate)s, and poly(phosphazene)s, have been studied for controlled drug delivery and tissue engineering, polylactide (PLA) and poly(lactide-co-glycolide) (PLGA) still remain the most popular and well characterized bio degradable polymeric biomaterials. Their regulatory approval and extensive database of human use render them obvious choice in contemplating a medical application that ranges from controlled drug delivery to tissue engineering.
The widening scope of applications in controlled delivery and tissue engineering require the biomaterials to assume different configurations to serve different functions. Applying the controlled release device as more than just a monolithic matrix, for example, as coating materials for a drug-eluting stent, may obligate the polymer to have elastomeric properties. In the new and exciting field of tissue engineering where local and sustained delivery of growth factors may influence the course of tissue development, the polymeric drug-carriers may also need to provide structural support or scaffolding functions. With such a broad utility for these biodegradable materials, PLA and PLGA cannot be expected to satisfy all requirements of different applications.
Besides physical blending, one of the most plausible ways to adjust the physico-chemical properties of PLA and PLGA is through copolymerization. Lactide copolymers with different constituent monomers can offer a broad range of physico-chemical properties and degradation rates. Poly(lactide-co-ester)s [e.g. (lactide-co-caprolactone)], poly(lactide-co-ether)s [e.g. poly(dioxanone), poly(ethylene glycol-b-lactide)], poly(lactide-co-carbonate) [e.g. poly(lactide-co-1,3-dioxan-2-one)], and poly(lactide-co-amide)s [poly(lactide-co-L-lysine)] are a few of the examples that have been evaluated so far.
Mao et al. (Mao, H.-Q., Z. Zhao, J. P. English and K. W. Leong (1997), Biodegradable polymers chain-extended byphosphoesters, compositions, articles and methods for making and using the same. U.S. Pat. No. 6,166,173) synthesized oligomeric lactide chain extended with alkyl phosphate in step growth polymerization processes. The resulting polymers had a more linear in vitro and in vivo degradation, compared with PLA with a similar molecular weight. However, compositions could only be prepared with a narrow range of phosphate incorporation, limiting the extent to which physical properties of the composition could be modified. The overall physico-chemical properties were similar to PLA/PLGA.
Fan et al. (Fan, C.-L., B. Li, Z.-H. Liu, R.-X. Zhuo (1995), A study on ring-opening copolymerization of D,L-lactide and 2-hydro-2-oxo-1,3,2-dioxaphosphorinane, Chemical Journal of Chinese Universities, 16(6), 971-973) have studied the random polymerization of D,L-lactide and 2-hydro-2-oxo-1,3,2-dioxaphosphorinane in the presence of triisobutylaluminum. The resulting copolymers are crosslinked, resulting in poor polymer solubility in both organic and aqueous solvents. 5-Fluorouracil (5-FU) was conjugated to the side chain of phosphite group. The release of 5-FU was close to first order release (Fan, C.-L., B. Li, Z.-H. Liu, R.-X. Zhuo (1996), Studies on synthesis and controlled release of polymeric drug using lactide-phosphate copolymer as drug carriers, Chemical Journal of Chinese Universities, 17(11), 1788-1791). The poor solubility of these polymers make them unsuitable for use in the present invention.
The present invention is directed to a series of biodegradable, biocompatible polymers comprising repeat units derived from cyclic phosphate monomers, e.g., ethylene methyl phosphate and the like, and optionally comprising repeat units derived from functionalized lactones and functionalized glycolide derivatives, e.g., (L,L)-lactide, (D,D)-lactide, meso-lactide, mixtures thereof and the like. The polymers of the invention have unique structures and physico-chemical properties.
Polymers of the invention comprise phosphoester backbone linkages which offers several advantages including (1) adjustable properties because the structures of both the backbone and the side chain can be varied; (2) lower glass transition temperature end better solvent solubility because of the plasticizing effect of the phosphate bond; (3) a natural candidate for a biodegradable pendant delivery system because of the availability of a functional side chain; (4) potentially nontoxic breakdown products because of a backbone analogous to nucleic acid and teichoic acid. Copolymerization between lactide and phosphate brings hydrophilicity, flexibility, and the ability of side chain modification to PLA and PLGA systems.
The present invention features biodegradable polymers, the polymers comprise at least one repeat unit according to formula A: 
wherein
R is hydrogen, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl or optionally substituted cycloalkyl preferably optionally substituted C5-8-cycloalkyl;
R1 and R2 are each independently chosen from the group consisting of H, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl and optionally substituted alkoxy preferably optionally substituted C1-12-alkoxy;
x is 2, 3, or 4; and
at least one repeat unit selected from the group consisting of optionally substituted alkyl preferably optionally substituted C1-20-alkyl, optionally substituted alkenyl preferably optionally substituted C2-20-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-20-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl, xe2x80x94O(CR1R2)cC(O)xe2x80x94 where c is between about 1 and about 10, xe2x80x94(CH2)axe2x80x94{O(CH2)a}bxe2x80x94 where a is between about 1 and about 7 and b is between about 1 and about 500, optionally substituted aryl, and optionally substituted heteroaryl; or
at least one repeat unit according to formula B: 
wherein
R3, R4, R5 and R6 are each independently chosen from the group consisting of H, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl and optionally substituted alkoxy preferably optionally substituted C1-12-alkoxy.
The biodegradable polymers of the present invention are biocompatible before and upon biodegradation.
Preferred polymers of the invention can be either linear or branched and are preferably substantially free of inter-polymer chain crosslinking.
Preferred polymers of the invention comprise either lactide derived repeat units according to formula B: 
wherein
R3, R4, R5 and R6 are each independently chosen from the group consisting of H, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl, and optionally substituted alkoxy preferably optionally substituted C1-12-alkoxy;
or caprolactone derived repeat units, e.g., xe2x80x94O(CR1R2)cC(O)xe2x80x94 where c is between about 1 and about 10, preferably c is between about 3 and about 6 more preferably c is about 5.
The biodegradable polymers of the invention exhibit unique physiochemical properties when compared to PLA and PLGA.
The present invention also features a process for preparing a biodegradable polymer comprising the recurring monomeric units of formula I: 
wherein:
R is hydrogen, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl or optionally substituted cycloalkyl preferably optionally substituted C5-8-cycloalkyl;
R1 and R2 are each independently chosen from the group consisting of H, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl and optionally substituted C1-12-alkoxy;
x is 2, 3, or 4;
n and m are non-negative integers;
n+m is about 5 to about 2000; and
m:n is between about 1:100 to about 100:1;
wherein the biodegradable polymer is biocompatible before and upon biodegradation processes occur, the process comprising the steps of:
contacting a glycolide derivative and a cyclic phosphate under conditions conducive to the formation of a biodegradable polymer comprising repeat units originating from the glycolide derivative and the cyclic phosphate.
The present invention also features a process for preparing a biodegradable polymer comprising the recurring monomeric units of formula III: 
wherein:
R is hydrogen, optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl or optionally substituted alkyl preferably optionally substituted C1-12-alkyl;
R1 and R2 are each independently chosen at each occurrence from the group consisting of hydrogen, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl and optionally substituted alkoxy preferably optionally substituted C1-12-alkoxy;
L is chosen from the group consisting of optionally substituted alkyl preferably optionally substituted C2-20-alkyl, optionally substituted alkenyl preferably optionally substituted C2-20-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-20-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl or xe2x80x94(CH2)axe2x80x94{Oxe2x80x94(CH2)a}bxe2x80x94, wherein each a is 1-6 and b is 1-500;
n and m are non-negative integers;
n+m is about 5 to about 2000;
x is 2, 3 or 4; and
M is independently chosen at each occurrence of M from the group consisting of H, Na, Li, and K;
the process comprising the steps of:
contacting at least one cyclic phosphate with an initiator compound, HOxe2x80x94Lxe2x80x94OH;
polymerizing the cyclic phosphate with the initiator compound under conditions conducive to preparing the biodegradable polymer.
The present invention also features a process for preparing a biodegradable polymer comprising the recurring monomeric repeat units of formula V: 
wherein
L is optionally substituted alkyl preferably optionally substituted C1-20-alkyl, optionally substituted alkenyl preferably optionally substituted C2-20-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-20-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-20-heteroalkyl, xe2x80x94(CH2)axe2x80x94{O(CH2)a}b where a is between about 1 and about 7 and b is between about 1 and about 500, optionally substituted aryl, or optionally substituted heteroaryl;
x is 2, 3, or 4;
R is hydrogen, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-20-heteroalkyl, or optionally substituted cycloalkyl preferably optionally substituted C5-8-cycloalkyl;
R1 and R2 are each independently chosen from the group consisting of hydrogen, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl, and optionally substituted alkoxy preferably optionally substituted C1-12-alkoxy;
n and m are non-negative integers;
n+m is about 5 to about 2000;
m:n is between about 1:100 to about 100:1;
c and d are non-negative integers;
c+d is about 5 to about 2000;
(m+n):(c+d) is between about 1:100 and 100:1; and,
the process comprising the steps of:
making at least one biodegradable polymer of claim 13;
contacting the biodegradable polymer of claim 13 with at least one glycolide derivative; and
polymerizing the glycolide derivative under conditions conducive to the preparing a biodegradable polymer according to formula V.
The present invention also features biodegradable polymer composition comprising:
(a) at least one biologically active substance; and
(b) a biodegradable polymer comprising at least one repeat unit according to formula A: 
wherein
R is hydrogen, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl or optionally substituted cycloalkyl preferably optionally substituted C5-8-cycloalkyl;
R1 and R2 are each independently chosen from the group consisting of H, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl and optionally substituted alkoxy preferably optionally substituted C1-12-alkoxy;
x is 2, 3, or 4; and
at least one repeat unit selected from the group consisting of optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-20-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-20-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl, xe2x80x94O(CR1R2)cC(O)xe2x80x94 where c is between about 1 and about 10, xe2x80x94(CH2)axe2x80x94{O(CH2)a}bxe2x80x94 where a is between about 1 and about 7 and b is between about 1 and about 500, optionally substituted aryl, and optionally substituted heteroaryl; or
at least one repeat unit according to formula B: 
wherein
R3, R4, R5 and R6 are each independently chosen from the group consisting of H, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl and optionally substituted alkoxy preferably optionally substituted C1-12-alkoxy.
The present invention also features an article useful for implantation, injection, or otherwise being placed totally or partially within a body, the article comprising a biodegradable polymer composition comprising:
(a) at least one biologically active substance; and
(b) a biodegradable polymer comprising at least one repeat unit according to formula A: 
wherein
R is hydrogen, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl or optionally substituted cycloalkyl preferably optionally substituted C5-8-cycloalkyl;
R1 and R2 are each independently chosen from the group consisting of H, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl and optionally substituted alkoxy preferably optionally substituted C1-12-alkoxy;
x is 2, 3, or 4; and
at least one repeat unit selected from the group consisting of optionally substituted alkyl preferably optionally substituted C1-20-alkyl, optionally substituted alkenyl preferably optionally substituted C2-20-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-20-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl, xe2x80x94O(CR1R2)cC(O)xe2x80x94 where c is between about 1 and about 10, xe2x80x94(CH2)axe2x80x94{O(CH2)a}bxe2x80x94 where a is between about 1 and about 7 and b is between about 1 and about 500, optionally substituted aryl, and optionally substituted heteroaryl; or
at least one repeat unit according to formula B: 
wherein
R3, R4, R5 and R6 are each independently chosen from the group consisting of H, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl and optionally substituted alkoxy preferably optionally substituted C1-12-alkoxy.
In preferred embodiments the biodegradable polymer is biocompatible before and upon biodegradation
The present invention also features a method for the controlled release of at least one biologically active substance comprising the steps of:
(a) combining the biologically active substance with a biodegradable polymer comprising at least one repeat unit according to formula A: 
wherein
R is hydrogen, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl or optionally substituted cycloalkyl preferably optionally substituted C5-8-cycloalkyl;
R1 and R2 are each independently chosen from the group consisting of H, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl and optionally substituted alkoxy preferably optionally substituted C1-12-alkoxy;
x is 2, 3, or 4; and
at least one repeat unit selected from the group consisting of optionally substituted alkyl preferably optionally substituted C1-20-alkyl, optionally substituted alkenyl preferably optionally substituted C2-20-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-20-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl, xe2x80x94O(CR1R2)cC(O)xe2x80x94 where c is between about 1 and about 10, xe2x80x94(CH2)axe2x80x94{O(CH2)a}bxe2x80x94 where a is between about 1 and about 7 and b is between about 1 and about 500, optionally substituted aryl, and optionally substituted heteroaryl; or
at least one repeat unit according to formula B: 
wherein
R3, R4, R5 and R6 are each independently chosen from the group consisting of H, optionally substituted alkyl preferably optionally substituted C1-12-alkyl, optionally substituted alkenyl preferably optionally substituted C2-12-alkenyl, optionally substituted alkynyl preferably optionally substituted C2-12-alkynyl, optionally substituted heteroalkyl preferably optionally substituted C1-12-heteroalkyl and optionally substituted alkoxy preferably optionally substituted C1-12-alkoxy;
to form an admixture;
(b) forming said admixure into a shaped, solid article or microsphere; and
(c) implanting or injecting the solid article or microsphere in vivo at a preselected site, such that the solid implanted or injected matrix is in at least partial contact with a biological fluid.
U.S. Pat. Nos. 5,912,225 entitled xe2x80x9cBiodegradable Poly(phosphoester-Co-Desaminotyrosyl L-Tyrosine Ester) Compounds, Compositions, Articles and Methods for Making and Using the Samexe2x80x9d and U.S. Pat. No. 6,166,173 entitled xe2x80x9cBiodegradable Polymers Chain Extended by Phosphates, Compositions, Articles and Methods for Making and Using the Samexe2x80x9d are incorporated herein by reference in their entirety.